CN103232983A - Culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.) - Google Patents
Culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.) Download PDFInfo
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Abstract
The invention relates to a culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.). The culture medium is suitable for the growth of the deep marine geobacillus sp (Geobacillus sp.) and can greatly increase the output of the amylases.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to produce diastatic substratum and preparation method thereof for ocean, deep-sea thermophilic bacterium Geobacillus sp. (more preferably being Geobacillus sp.4j).
Background technology
Thermophilic marine microorganism is a class thermophilic microorganism resource that derives from particular surroundingss such as underground heat oil-containing geological stratification, deep-sea volcanic mud, deep-sea hydrothermal port.Thermophilic microorganism is because its adaptability to the high temperature uniqueness enjoys people to pay close attention to, and refers to that generally optimum growth temperature is greater than a quasi-microorganism of 45 ℃.As thermophilic microorganism, it one of is mainly used is the Application and Development of Zimadzhunt L 340.Zimadzhunt L 340 gets most of the attention because of the high advantage of its thermostability, and wherein the most famous is exactly discovery and the exploitation of Tag enzyme, and making PCR react this revolutionary technical development becomes one of of paramount importance technology of life science.At present the known enzyme tool high thermal stability of from thermophile bacteria, separating, rate of mass transfer height, chemically-resistant denaturing agent, be easy to characteristics such as purifying.
Ocean, deep-sea thermophilic bacterium Geobacillus sp.4j is by being to be separated first by Chinese ocean the 3rd institute to obtain, derive from deep-sea, optimum growth temperature at a kind of moderate thermophilic microorganism of 60 ℃.Because it derives from this extreme environment of deep-sea, it is corresponding to have special physiology to envrionment conditionss such as dissolved oxygen, high salt, high temperature, high pressure.In application facet, the present discovery of Geobacillus sp.4j can produce the amylase of degraded starch.Can the diastatic activity of this kind and thermostability etc. remain further to be explored, also be worth research and be used for a kind of amylase of suitability for industrialized production.
Yet the thermophile bacteria Geobacillus sp.4j diastatic output of growing in original substratum is very low, becomes the bottleneck of every research, produces the field of enzyme especially in its mass-producing fermentation culture.If can not improve diastatic output, just be difficult to carry out the exploration of corresponding enzymology and suitability for industrialized production.
Therefore, by optimizing its culture environment, improving its zymotechnique and become the task of top priority.Substratum is the matrix of microorganism growth, develops that a kind of to produce diastatic novel culture medium for ocean, deep-sea thermophilic bacterium Geobacillus sp.4j be the present important foundation of work.
Summary of the invention
The object of the present invention is to provide for ocean, deep-sea thermophilic bacterium Geobacillus sp. and produce diastatic substratum, its preparation method and application.
In a first aspect of the present invention, the substratum of ocean, a kind of deep-sea thermophilic bacterium Geobacillus sp. is provided, described substratum comprises following component:
In a preference, ocean, described deep-sea thermophilic bacterium Geobacillus sp. is Geobacillus sp.4j.
In another preference, described substratum comprises following component:
In another preference, described substratum comprises following component:
Wherein, the content of each component in substratum can float in ± 10% (more preferably ± 5%) scope.
In another preference, in the described substratum, also contain the trace element of significant quantity (being suitable for the amount of bacterial growth); Preferably, micronutrient levels is:
In another preference, in the described substratum, each set of dispense is formed in the water (preferably, described water is deionized water); And/or the pH value 6 ± 0.5 of described substratum.
In another aspect of this invention, provide the purposes of the arbitrary described substratum in front, be used for cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp., produce amylase.
In another aspect of this invention, provide a kind of method for preparing the substratum of ocean, arbitrary described deep-sea, front thermophilic bacterium Geobacillus sp., described method comprises:
(a) described Zulkovsky starch is carried out gelatinization, cooling;
(b) described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate are mixed;
(c) solution that above-mentioned two steps are obtained mixes, and adds nickel chloride solution and optional trace element;
Wherein, the consumption of each component is arbitrary described according to the front.
In another preference, in step (a), described gelatinization is slowly poured 90-100 ℃ of deionized water and constantly stirring into then for less water (preferably being deionized water) described Zulkovsky starch being made suspension liquid, makes translucent liquid.
In another preference, step (c) further comprises afterwards: pH is transferred to 6 ± 0.5, at 121 ℃ of sterilization 20-30min.
In another aspect of this invention, provide a kind of ocean, deep-sea thermophilic bacterium Geobacillus sp. that utilizes to produce diastatic method, described method comprises: utilize ocean, arbitrary described culture medium culturing deep-sea, front thermophilic bacterium Geobacillus sp., thereby produce amylase.
In a preference, with ocean, deep-sea thermophilic bacterium Geobacillus sp. by volume the inoculum size of 1-5% inoculate into the arbitrary described substratum in front, 60 ℃, static condition bottom fermentation cultivation 72-96h, leave standstill in the culturing process and carry out oscillation treatment one time every 12h, will cultivate bacterium liquid mixing; After cultivating 72h and 96h, the centrifugation fermented supernatant fluid wherein contains amylase.
In another preference, be used for the OD of the seed liquor of inoculation
600Be 1.8-2.4, colony number density is about 2.1 * 10
6-2.8 * 10
6CFU/ml.
In another aspect of this invention, provide a kind of for the test kit of cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp., comprise in the described test kit: the arbitrary described substratum in front.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Embodiment
The inventor is through long-term and deep research, researched and developed a kind of new being suitable for and cultivated ocean, deep-sea thermophilic bacterium Geobacillus sp. (more preferably being Geobacillus sp.4j) and make it the diastatic culture medium prescription of High-efficient Production.Substratum of the present invention is suitable for the growth of ocean, deep-sea thermophilic bacterium Geobacillus sp., and can improve diastatic output widely.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
Term " substratum of ocean, deep-sea of the present invention thermophilic bacterium Geobacillus sp. " refers to contain the composition of Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element; Or the composition of being formed by Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element basically.In composition, described Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element account for the 80-100% of substratum gross weight, preferably account for 90-100%, more preferably account for 95-100%, as 98%, 99%.
As optimal way of the present invention, as shown in table 1 for the consumption of each component of preparing ocean, deep-sea of the present invention thermophilic bacterium Geobacillussp. (more preferably being Geobacillus sp.4j) substratum.
Table 1
As optimal way of the present invention, also add trace element in the described substratum, the addition of trace element is to satisfy thalli growth and to produce required being advisable, and those skilled in the art can add according to experience.As optimal way of the present invention, the consumption of each component of trace element is as shown in table 2.
Table 2
After learning ocean, deep-sea of the present invention thermophilic bacterium Geobacillus sp. (more preferably being Geobacillus sp.4j) substratum component utilized and prescription thereof, those skilled in the art can prepare easily.
The component of the application of substratum of the present invention (raw material) is business-like general chemistry product, price comparison is low, prepare also very simple, compare with the substratum of prior art, novel culture medium of the present invention can significantly promote ocean, deep-sea thermophilic bacterium Geobacillus sp. fermentative production amylase, diastatic output is significantly improved, and is conducive to large-scale industrial production.
As optimal way of the present invention, when preparation ocean, described deep-sea thermophilic bacterium Geobacillus sp. (more preferably being Geobacillus sp.4j) substratum, accurately take by weighing each component respectively earlier, they are mixed in the water.Described water preferably is deionized water.Preferred, a kind of method for the preparation of above-mentioned substratum is provided, comprise step:
(a) described Zulkovsky starch is carried out gelatinization, cooling;
(b) described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate are mixed;
(c) solution that above-mentioned two steps are obtained mixes, and adds nickel chloride solution and optional trace element.
Described Zulkovsky starch need carry out gelatinization earlier to be handled, and purpose is to prevent the problem of luming in the sterilization process.The gelatinization of Zulkovsky starch can be adopted the known any method of those skilled in the art.As optimal way of the present invention, described gelatinization is slowly poured 90-100 ℃ of deionized water and constantly stirring into then for less water (preferably being deionized water) described Zulkovsky starch being made suspension liquid, makes translucent liquid.
As optimal way of the present invention, step (c) further comprises afterwards: pH is transferred to 6 ± 0.5, at 121 ℃ of sterilization 20-30min.
The present invention also provides and has used ocean, described culture medium culturing deep-sea thermophilic bacterium Geobacillus sp. (more preferably being Geobacillus sp.4j) the diastatic fermentation process of fermentative production, comprise step: utilize ocean, described culture medium culturing deep-sea thermophilic bacterium Geobacillus sp., thereby produce amylase.
The diastatic detection method that the present invention also provides a kind of above-mentioned fermentation process to produce, its operation steps comprises:
A. with rifle head picking list bacterium colony from the solid plate of fresh Geobacillus sp.1j of inoculating needle or sterilization, be seeded to the 250ml that 80ml activated seed substratum is housed and shake in the bottle, 60 ℃, 100rpm shaker fermentation cultivation 16-20h obtain fresh seeds liquid;
B. get described fresh seeds liquid, the 250ml that the described novel culture medium of 50ml is housed by the inoculum size access of 1% (v/v) shakes in the bottle, the silica gel plug sealing, 60 ℃, be statically placed in fermentation culture 72-96h in the high temperature constant temperature shaking table, for the whole culturing process that leaves standstill, carry out 15min one time every 12h, the vibration of 100rpm will be cultivated bacterium liquid mixing, and bacterium liquid is mixed.After cultivating 72h and 96h, 12000rpm, 4 ℃ of centrifugal 3min obtain fermented supernatant fluid and are used for the mensuration that enzyme is lived, and get wherein bigger value.
Preferably, after the bacterial classification of-80 ℃ of preservations thawed, get 1 ring bacterium liquid with the aseptic inoculation ring, rule at the solid plate activation medium, place 60 ℃ of constant incubators, activation culture 8-12h obtains fresh solid plate, place 4 ℃ of refrigerators stand-by then, later solid plate reactivated once every 30 days.
Preferably, every liter of activation medium includes yeast powder 2.5g, peptone 2.5g, nitrilotriacetic acid(NTA) 0.1g, terra alba 0.04g, magnesium chloride 0.2g, ironic citrate 0.01675g, liquid microelement I0.5ml, phosphate buffer 1 00ml, all the other are deionized water, and pH transfers to about 7.35, at 121 ℃ of sterilization 20-30min.
Preferably, every liter of solid plate activation medium is to add agar powder 15-20g on the basis of activation medium.
Preferably, the OD of fresh seed liquor fresh seeds liquid during inoculation
600Be 1.8-2.4, colony number density is about 2.1 * 10
6-2.8 * 10
6CFU/ml.
Preferably, contain dipotassium hydrogen phosphate 5.44g in every liter of phosphate buffered saline buffer, disodium hydrogen phosphate dodecahydrate 43g, all the other are deionized water, pH transfers to about 7.2.
Medium component of the present invention is clear and definite, addition is low, with low cost, preparation manipulation is convenient, can significantly improve ocean, deep-sea thermophilic bacterium Geobacillus sp.4j and under hot environment, grow and produce heat-resisting diastatic output, make that the diastatic enzyme work in the fermented supernatant fluid is brought up to 6.36U/ml by original 2U/ml.This produces the fermentation research of enzyme for ocean, deep-sea thermophilic bacterium and novel diastatic exploitation has great importance.
The present invention also provides a kind of diastatic detection method of utilizing described fermentation process to obtain, comprises step: get some test tube marks, draw an amount of 1% starch substrates (pH7.50) with pipettor, put into 60 ℃ of water bath with thermostatic control preheating 10min.The fermented supernatant fluid that an amount of described fermentation process is obtained adds in the test tube then, and mixing accurately reacts for some time immediately.After the reaction, reaction tube is transferred in the ice-water bath immediately, added appropriate amount of deionized water and an amount of DNS reagent immediately, mix.After being transferred to 100 ℃ of accurate clock reaction 5min of water bath with thermostatic control then, putting into mixture of ice and water immediately cools off, add the certain volume deionized water then, mixing is used the absorbance A at spectrophotometric determination 540nm place again, according to the reducing sugar content in the light absorption value A conversion response liquid.Measure the reducing sugar content that just had originally in starch substrates and the fermented supernatant fluid then simultaneously, can calculate diastatic enzyme work in the described fermented supernatant fluid by calculating corresponding difference.Wherein 1 enzyme unit definition alive is the enzyme amount that the per minute catalytic decomposition produces 1 micromole's reducing sugar under the above-mentioned condition.
Beneficial effect of the present invention is specific as follows:
1. novel culture medium composition of the present invention comprises common reagent such as Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, the liquid microelement addition is low, raw material is easy to get, and consumption is few, and cost is low;
2. when preparing novel culture medium of the present invention, earlier starch is made suspension with deionized water, boil gelatinization, cooling again, in deionized water, both mix, and add a small amount of liquid microelement again with all the other components dissolved, and constant volume get final product, and preparation is simply;
3. adopt novel culture medium of the present invention to cultivate ocean, deep-sea thermophilic bacterium Geobacillus sp.4j, this bacterial strain diastatic output of growing in this novel culture medium is significantly increased, and makes that the diastatic enzyme work in the fermented supernatant fluid is brought up to 6.36U/ml by original 2U/ml.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Sa nurse Brooker etc. usually, molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
In addition, adopt the equipment such as high temperature constant temperature shaking table of different manufacturers, may cause a little difference of experimental result, suchlike difference belongs to normal phenomenon.
Embodiment 1, substratum 1
1) Zulkovsky starch gelatinization
Take by weighing the 10g Zulkovsky starch, make suspension liquid with the 20ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take by weighing yeast powder 4g, SODIUMNITRATE 3g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 8.92g, Repone K 0.33g, calcium chloride 0.1g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour step 2 again into) the cooled starch dextrin of preparation, add liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 6 with deionized water again.
Wherein preparing liquid microelement I is prepared as follows:
Nitrilotriacetic acid(NTA) 12.8g, Iron dichloride tetrahydrate 1g, four hydration Manganous chloride tetrahydrate 0.5g, four hydrated cobalt chloride 0.3g, Copper dichloride dihydrate 0.05g, Sodium orthomolybdate 0.05g, boric acid 0.02g, Nickel dichloride hexahydrate 0.02g, all the other are deionized water, are settled to 1000ml, and pH transfers to 6.8.Long-time storage needs to use the needle-based strainer filtration sterilization of 0.22 μ m.
3) fermentation culture process
Ocean, deep-sea thermophilic bacterium Geobacillus sp.4j is provided by Chinese ocean the 3rd institute.Picking list bacterium colony from activated good solid plate is seeded to the 250ml that 80ml activated seed substratum is housed and shakes in the bottle, and 60 ℃, 100rpm shaker fermentation cultivation 18h obtain fresh seeds liquid; The OD of fresh seeds liquid
600Be that 2.1 (colony number density is about 2.4 * 10
6CFU/ml), by the inoculum size of 1% (v/v) fresh seeds liquid being inserted the 250ml that the described novel culture medium of 50ml is housed shakes in the bottle, 60 ℃, be statically placed in fermentation culture 96h in the high temperature constant temperature shaking table, for the whole culturing process that leaves standstill, carry out 15min, the vibration of 100rpm one time every 12h, to cultivate bacterium liquid mixing, bacterium liquid is mixed.After cultivating 96h, 12000rpm, 4 ℃ of centrifugal 3min obtain fermented supernatant fluid and are used for the mensuration that enzyme is lived.
Wherein, every liter of activation medium includes yeast powder 2.5g, peptone 2.5g, nitrilotriacetic acid(NTA) 0.1g, terra alba 0.04g, magnesium chloride 0.2g, ironic citrate 0.01675g, liquid microelement I0.5ml, phosphate buffer 1 00ml, all the other are deionized water, and pH transfers to about 7.35, at 121 ℃ of sterilization 20-30min.Contain dipotassium hydrogen phosphate 5.44g in every liter of phosphate buffered saline buffer, disodium hydrogen phosphate dodecahydrate 43g, all the other are deionized water, pH transfers to about 7.2.
4) diastatic enzyme activity determination
Get some test tube marks, (adopt Sodium phosphate dibasic-citrate buffer solution gelatinization and preparation of 0.05M, pH7.50) 280 μ L put into 60 ℃ of water bath with thermostatic control preheating 10min to the starch substrates of absorption 10g/L gelatinization in advance.Then fermented supernatant fluid 20 μ L are added in the test tube, mixing accurately reacts 10min immediately.After the reaction, reaction tube is transferred in the ice-water bath immediately, added the DNS reagent of 200 μ L deionized waters and 300 μ L immediately, mix.After being transferred to 100 ℃ of accurate clock reaction 5min of water bath with thermostatic control then, putting into mixture of ice and water immediately cools off, add ionized water 6ml then, mixing is used the absorbance A at spectrophotometric determination 540nm place again, according to the reducing sugar content in the light absorption value A conversion response liquid.Measure in starch substrates and the fermented supernatant fluid reducing sugar content that just had originally then simultaneously, can calculate and produce the diastatic enzyme that the content of reducing sugar characterizes with the per minute hydrolysis and live, measurement result, enzyme is lived and is 4.4U/ml.
Embodiment 2, substratum 2
1) Zulkovsky starch gelatinization
The 10g Zulkovsky starch is made suspension liquid with the 20ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take by weighing yeast powder 8g, SODIUMNITRATE 1g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 15g, Repone K 0.33g, calcium chloride 0.15g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour the cooled starch dextrin of step 1) preparation again into, add liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 6 with deionized water again.
3) fermentation culture process
Step 3) with embodiment 1.
4) fermentation culture is measured
With the step 4) of embodiment 1, measure the diastatic enzyme 4.98U/ml of being alive.
Embodiment 3, substratum 3
1) Zulkovsky starch gelatinization
Take by weighing the 9g Zulkovsky starch and make suspension liquid with the 20ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take by weighing yeast powder 8g, SODIUMNITRATE 7.5g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 6g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.2g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour the cooled starch dextrin of step 1) preparation again into, add liquid microelement I12ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 6 with deionized water again.
3) fermentation culture process
Step 3) with embodiment 1.
4) fermentation culture is measured
With the step 4) of embodiment 1, measure the diastatic enzyme 5.08U/ml of being alive.
Embodiment 4, substratum 4
1) Zulkovsky starch gelatinization
Take by weighing the 6g Zulkovsky starch and make suspension liquid with the 15ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, make translucent uniform viscous liquid.
2) substratum preparation
Take by weighing yeast powder 8g, SODIUMNITRATE 5g, sodium-chlor 15g, anhydrous magnesium sulfate 1.67g, magnesium chloride 2g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.4g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour the cooled starch dextrin of step 1) preparation again into, add liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 6 with deionized water again.
3) fermentation culture process
For ease of relatively, with the step 3) of embodiment 1.
4) cell density is measured
With the step 4) of embodiment 1, measure the diastatic enzyme 6.2U/ml of being alive.
Embodiment 5, substratum 5
1) Zulkovsky starch gelatinization
Take by weighing the 6g Zulkovsky starch and make suspension liquid with the 12ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, to becoming translucent uniform viscous liquid, cooling.
2) substratum preparation
Take by weighing yeast powder 8g, SODIUMNITRATE 5g, sodium-chlor 12.5g, anhydrous magnesium sulfate 1.67g, magnesium chloride 0.32g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.484g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour the cooled starch dextrin of step 1) preparation again into, add liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 6 with deionized water again.
3) fermentation culture process
Step 3) with embodiment 1.
4) cell density is measured
With the step 4) of embodiment 1, measure the diastatic enzyme 6.36U/ml of being alive.
Embodiment 6, control group example
1) Zulkovsky starch gelatinization
Take by weighing the 30g Zulkovsky starch and make suspension liquid with the 50ml deionized water, slowly pour into then in the deionized water that has just boiled and constantly and stir, to becoming translucent uniform viscous liquid, cooling.
2) substratum preparation
Take by weighing yeast powder 5g, peptone 5g, sodium-chlor 30g, anhydrous magnesium sulfate 1.67g, magnesium chloride 8.92g, Repone K 0.33g, calcium chloride 0.1g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pouring appropriate amount of deionized water into fully dissolves mentioned component, pour the cooled starch dextrin of step 1) preparation again into, add liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, be settled to 1000ml, pH regulator to 7.35 with deionized water again.
3) fermentation culture process
Step 3) with embodiment 1.
4) cell density is measured
With the step 4) of embodiment 1, measure the diastatic enzyme 2U/ml of being alive.
Can find from embodiment 1～6: utilize novel culture medium of the present invention and preparation method, the diastatic enzyme work that makes ocean, deep-sea thermophilic bacterium Geobacillus sp.4j produce in the diastatic fermented supernatant fluid of hot temperature is brought up to more than the 4U/ml by in the past 2U/ml, more can reach 6.36U/ml.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the substratum of ocean, deep-sea thermophilic bacterium Geobacillus sp. is characterized in that described substratum comprises following component:
2. substratum as claimed in claim 1 is characterized in that, described substratum comprises following component:
3. substratum as claimed in claim 2 is characterized in that, described substratum comprises following component:
Wherein, the content of each component in substratum can float in ± 10% scope.
4. as the arbitrary described substratum of claim 1-3, it is characterized in that, in the described substratum, also contain the trace element of significant quantity; Preferably, micronutrient levels is:
5. as the arbitrary described substratum of claim 1-4, it is characterized in that in the described substratum, each set of dispense is formed in the water; And/or
The pH value 6 ± 0.5 of described substratum.
6. the purposes of the arbitrary described substratum of claim 1-5 is used for cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp., produces amylase.
7. method for preparing the substratum of ocean, the arbitrary described deep-sea of claim 1-5 thermophilic bacterium Geobacillus sp. is characterized in that described method comprises:
(a) described Zulkovsky starch is carried out gelatinization, cooling;
(b) described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate are mixed;
(c) solution that above-mentioned two steps are obtained mixes, and adds nickel chloride solution and optional trace element;
Wherein, the consumption of each component is arbitrary described according to claim 1-5.
8. one kind is utilized ocean, deep-sea thermophilic bacterium Geobacillus sp. to produce diastatic method, it is characterized in that, described method comprises: utilize ocean, the arbitrary described culture medium culturing deep-sea of claim 1-5 thermophilic bacterium Geobacillus sp., thereby produce amylase.
9. method as claimed in claim 8, it is characterized in that, with ocean, deep-sea thermophilic bacterium Geobacillus sp. by volume the inoculum size of 1-5% inoculate arbitrary described substratum into claim 1-5,60 ℃, static condition bottom fermentation cultivation 72-96h, leave standstill in the culturing process and carry out oscillation treatment one time every 12h, will cultivate bacterium liquid mixing; After cultivating 72h and 96h, the centrifugation fermented supernatant fluid wherein contains amylase.
10. a test kit that is used for cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp. is characterized in that, comprises in the described test kit: the arbitrary described substratum of claim 1-5.
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| CN104560914A (en) * | 2014-12-29 | 2015-04-29 | 国家海洋局第三海洋研究所 | Alpha-amylase Amy16, as well as gene and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560914A (en) * | 2014-12-29 | 2015-04-29 | 国家海洋局第三海洋研究所 | Alpha-amylase Amy16, as well as gene and application thereof |
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| CN103232983B (en) | 2015-03-11 |
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